APL is associated with reciprocal translocations always involving the RARalpha locus that variably fuses to the PML, PLZF, NPM, NuMA or STAT5b genes (referred to as X genes). Two aberrant fusion genes are thus generated which encode X-RARalpha and RARalpha-X fusion proteins. We hypothesize that the fusion proteins act to disrupt/interfere with the normal biological function of X and RARa proteins, and that additional genetic events synergistically cooperate with X-RARalpha and RARalpha-X leading to APL. We will test this hypothesis in vivo, by a direct genetic approach, with the following Specific Aims: 1) To identify and characterize additional genetic events that contribute to APL pathogenesis as well as target genes of the various fusion proteins. Retroviral insertional mutagenesis in XRARalpha/ RARalpha-X TM, Spectral Karyotyping (SKY) and ChIP chip technology will be utilized to identify additional genetic events and target genes in APL leukemogenesis. 2) To assess in a high-throughput manner the functional significance of cooperative events and target genes in APL leukemogenesis, and the role of the fusion protein in leukemia maintenance, by utilizing a TVA-based approach. The TVA system, which we have optimized for the analysis of cooperative leukemogenesis, will be utilized to study the contribution of additional genetic events and deregulated gene expression in APL pathogenesis. The role of the X-RARalpha fusion protein in the maintenance of the leukemic phenotype upon leukemia development and treatment will also be assessed taking advantage of a combined TVA-tetracycline inducible system. 3) To determine the consequence of an impairment in TGF-beta signaling in APL leukemogenesis. We have shown that PML-RARalpha can disrupt the essential role of cytoplasmic PML isoforms in TGF-beta signaling. We will study whether and through which mechanisms the other X-RARalpha fusion proteins of APL exert a similar function. Through an in vivo genetic approach in the mouse we will determine the relevance of TGF-beta blockade in APL leukemogenesis.4) To define the role of HIF-1alpha and its downstream target genes in APL leukemogenesis. We will study the mechanisms underlying aberrant VEGF upregulation by PML-RARalpha and its pathophysiological consequences both in vitro and in vivo in APL mouse models. We will assess the importance of neoangiogenesis in APL leukemogenesis in PML-RARalpha/Id1+/-Id3-/- compound mutants. We will determine whether blockade of the Hif-1alpha/VEGF pathway can be exploited for APL therapy in preclinical trials. 5) To assess the contribution of centrosome amplification and genomic instability in APL leukemogenesis. We propose to study the mechanism by which NPM-RARalpha leads to centrosomal defects and whether the other X-RARalpha fusion proteins of APL share this critical activity. We will determine in vivo the relevance of aberrant centrosome duplication and genetic instability in APL leukemogenesis.